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Abstract

Introduction

Matrix metalloproteinases (MMPs) are implicated in the destruction of the joint and
have been shown to be strongly associated with inflammation in rheumatoid arthritis
(RA). Circulating MMPs have also been associated with cardiovascular disease in the
general population, and are predictive of cardiovascular mortality. The purpose of
the present study was to determine whether circulating levels of MMPs are predictive
of mortality in RA.

Methods

A multiplex suspension array system (Luminex®) was used to measure levels of MMPs (1, 2, 3, 8 and 9) in sera taken at recruitment
of RA patients (n = 487) in a study of factors associated with mortality in RA. Patients were tracked
on the National Health Service Central Register for notification of death, and the
relationship between baseline MMP levels and mortality was analysed using Cox proportional
hazards regression analysis.

Results

At the time of follow-up, 204/486 patients had died, of which 94 (46.1%) had died
of circulatory diseases, 49 of malignancy (24.0%), and 42 (20.6%) of respiratory diseases.
In a stepwise analysis which included all MMPs, only MMP-8 was significantly associated
with all cause mortality (P = 0.0007, 0.6% hazard ratio increase per ng/ml). No association was found between
MMP levels and mortality due to circulatory disease or malignancy. However MMP-8 levels
were strongly associated with mortality due to respiratory disease (P < 0.0001, 1.3% hazard ratio increase per ng/ml). The association with respiratory
disease related mortality remained highly significant in multivariate models which
included smoking as well as markers of severity and disease activity such as rheumatoid
factor, nodular disease, and C-reactive protein (CRP).

Conclusions

The serum level of MMP-8 is a strong predictor of mortality in RA, especially that
due to respiratory disease. This finding is consistent with increased activation of
neutrophils in RA and identifies serum MMP-8 as a useful marker for increased risk
of premature death.

Introduction

Many studies have shown that the mortality rate is increased in patients with rheumatoid
arthritis (RA) compared with the general population [1-6]. Causes of death in RA are similar to those in the general population but there is
an increased risk of death due to cardiovascular disease (CVD), and an excess of deaths
due to infection, much of which is due to lower respiratory tract infections [1,2,7-12].

Markers of inflammation such as erythrocyte sedimentation rate (ESR), C-reactive protein
(CRP) and soluble tumour necrosis factor receptors have been shown to be predictive
of mortality in RA [2,3,5,7-9,13], but many other markers associated with the disease process have not been investigated.
Some of these may provide alternative or better predictors of early mortality. One
such group of markers are the matrix metalloproteinases (MMPs). These are implicated
in the destruction of the joint and have been shown to be strongly associated with
inflammation and disease activity in RA [14-18]. Circulating MMPs have also been associated with CVD and atherosclerotic plaque instability
in non-RA patients [19-25], and have been shown to be predictive of cardiovascular mortality [20,25].

We postulated that elevated levels of circulating MMPs in RA patients may be associated
not only with increased rheumatoid disease activity and severity, but may also predict
premature mortality. We have thus investigated the relationship of circulating levels
of MMPs with all-cause, and cause-specific mortality, and determined whether any relationship
between mortality and MMP levels was independent of traditional risk factors and other
markers of inflammation and disease severity.

Materials and methods

Study population

This was a follow up study of a cohort of RA patients from North Staffordshire, England,
recruited between 1993 and 1998 to investigate long-term outcome and mortality in
patients attending a hospital-based clinic at the Haywood Rheumatology Centre. Consecutive
cases were selected from clinics of confirmed RA patients who satisfied the 1987 American
College of Rheumatology criteria for RA [26]. Baseline assessments included the disability index of the Stanford health assessment
questionnaire (HAQ) [27], presence of erosions, CRP and ESR levels, IgM rheumatoid factor (RF), and presence
or absence of nodular disease. Age, sex, disease duration at recruitment, and history
of current or past cigarette smoking was also recorded. As an indication of pre-existing
CVD at the time of recruitment, patients were stratified according to whether or not
they were taking any drugs for cardiovascular problems. This included patients taking
drugs for cardiac and non-cardiac conditions, the latter including cerebrovascular
disease and peripheral vascular disease. Patients taking drugs for hypertension alone
were not included in this group. The study was approved by the North Staffordshire
local research ethics committee.

The treatment of patients at baseline reflected standard UK practice for management
of hospital-based RA patients as outlined in published guidelines [28]. Patients were receiving anti-inflammatory and/or anti-rheumatic therapy, with the
majority of patients (> 90%) being treated with one or more disease modifying anti-rheumatic
drugs (DMARDs). DMARDs were chosen according to physician's preference, using the
standard practice at the time of sequential monotherapy and combination therapy for
more severe disease. The most common combination was methotrexate and sulphasalazine.
Steroids and cytotoxic drugs such as azathioprine or cyclophosphamide were received
by a small minority of individuals (< 5%). A number of patients (8%) were treated
with anti-TNF agents during the latter years of the study (from 2002). These patients
all fulfilled the United Kingdom National Institute of Clinical Excellence (NICE)
criteria for use of anti-TNF therapy.

Survival follow up

All patients were registered on the NHS Central Register (NHSCR), a computerised registry
of the records of all patients registered with a general practitioner in England and
Wales. Access to this registry was obtained via the Office for National Statistics
(ONS), General Register Office, Southport, UK. Patients were tracked on the NHSCR,
and notification of patient deaths was obtained from the ONS within 1 month of death.
Causes of death were coded by the ONS, using the International Classification of Diseases
Ninth Revision (ICD-9) [29] up until 31 December 2000, and ICD-10 after this date. Patients were followed up
to 31 December 2011.

Multiplex MMP assays

Sera were separated from bloods collected in plain glass Becton Dickinson (BD) (Becton
Dickinson, Oxford, Oxfordshire, UK) Vacutainer® tubes at study entry. Bloods were allowed to clot for 60 minutes at room temperature
before serum separation and storage at -70o C until required. Measurement of MMP-1, 2, 3, 8 and 9 levels was performed on the
serum samples using Fluorokine MAP multiplex kits (R&D Systems, Minneapolis, MN, USA)
and was read on a Luminex® suspension array system (Bio-Plex 200™, Bio-Rad (Bio-Rad Laboratories, Hemel Hempstead,
Hertfordshire, UK)). Sample preparation and assay procedure were followed according
to the manufacturer's recommendation. Heteroblock (Omega Biologicals, Bozeman, MT,
USA) was added at a concentration of 150 ug/ml into the sample diluent prior to assay,
to block any non-specific binding to RF [30]. All samples were run in duplicate with the appropriate standards on 96-well microplates.
The limit of detection for each of the MMPs was as follows: MMP-1, 0.04 ng/ml; MMP-2,
0.03 ng/ml; MMP-3, 0.01 ng/ml; MMP-8, 0.04 ng/ml; MMP-9, 0.74 ng/ml. The intra- and
inter-assay coefficients of variation for each of the MMPs were between 1.5 and 9.2%,
and 6.2 and 15.2% respectively.

Statistical analysis

Spearman's rank correlation was used to assess the relationship between MMP levels
and measures of disease activity and severity. The Mann-Whitney U-test was used to determine baseline differences in MMP levels between patients with
and without nodular or erosive disease, between smokers and non-smokers and between
surviving and non-surviving patients. The association between serum MMP levels and
mortality risk was investigated using Cox proportional hazard regression analyses
adjusted for age, sex and disease duration at baseline. The time intervals for those
patients who were alive at the end of the study period and those who were lost to
follow up were censored. The censoring date was the date of the last hospital visit.
Since mortality data are recorded centrally in the UK, we were able to collect mortality
data on all patients entered into the study, and patients lost to follow up without
a documented death could be censored with some degree of certainty. Multivariate stepwise
models were used to assess the predictive value of MMP levels compared with other
potential baseline risk factors (such as, RF, nodules, CRP, ESR, HAQ, taking CVD drugs,
smoking status). Separate analyses were carried out on the risk of mortality related
to each of the major causes of death in RA, namely circulatory, malignant and respiratory
diseases. In these analyses the data on subjects who died of other causes, and the
data on those who were still alive at last follow up were censored.

Results

Characteristics of RA patients

The baseline clinical features of the cohort are summarised in Table 1. Of the 487 patients recruited there were 204 deaths (41.9%), of which 86 were men
and 118 were women. For patients who died the median survival time from study baseline
was 6.9 years (interquartile range (IQR) 3.6 to 12.1). For survivors the median follow
up period from baseline was 16.2 years (IQR 5.9 to 17.2).

Main causes of death

The three major causes of death were due to circulatory disease (94/204, 46.1%), neoplasia
(49/204, 24.0%) and respiratory diseases (42/204, 20.6%). Of the 94 patients who died
from circulatory disease, 62 were due to heart disease (mainly ischaemic heart disease,
congestive heart failure, or left ventricular failure), and 19 were due to cerebrovascular
disease. The main cause of respiratory disease related mortality was pneumonia (29/42).
In this group, pneumonia was recorded as the only cause of death, or the only cause
with RA as a contributing cause in 23/29 patients. Of the remaining six patients with
pneumonia, three also had longstanding chronic obstructive pulmonary disease (COPD),
one had pulmonary fibrosis, one had CVD and one was associated with sepsis.

Relationship between MMP levels and patient characteristics at baseline

There was a positive correlation between the levels of all the MMPs apart from MMP-2
(Additional file 1). Comparison of MMP levels in men and women showed a significantly higher median
level of MMP-3 (28.1 v 21.2 ng/ml, P = 0.00008), MMP-8 (23.37 v 17.96 ng/ml, P = 0.0008) and MMP-9 (435.5 v 363.5 ng/ml, P = 0.001) in men. The levels of MMP-1, MMP-2 and MMP-3 showed a significant increase
with age (Rs ≥ 0.106, P ≤ 0.01), independent of disease duration (Additional file 2). Most measures of disease activity and severity (ESR, CRP, HAQ, Larsen score, visual
analogue (VAS) pain score) were positively correlated with levels of all MMPs apart
from MMP-2 (Additional file 2). The latter showed a negative correlation with CRP and the VAS pain score. No correlation
was found between IgM RF titres and MMP levels. Patients with erosive disease had
significantly higher levels of MMP-3 than those with non-erosive disease (25.46 v
15.35 ng/ml, P = 0.006), but no difference was found for other MMPs. The association with MMP-3 remained
significant after adjusting for age, sex and disease duration (data not shown). No
difference in MMP levels was found between patients with or without nodular disease.
Patients who were taking one or more drugs for CVD at baseline had significantly higher
levels of MMP-2, MMP-8 and MMP-9 than patients not taking these drugs (146.3 v 131.1
ng/ml, P = 0.007, 26.67 v 20.32 ng/ml, P = 0.02, and 471.5 v 413.7, P = 0.049 respectively). However, in a stepwise analysis adjusted for age, sex and disease
duration, there was only an association with MMP-9 levels (data not shown). Patients
that had ever smoked also had higher levels of MMP-8 and MMP-9 than those who had
never smoked (22.05 v 17.46 ng/ml, P = 0.015, and 411.2 v 344.8 ng/ml, P = 0.004 respectively).

Relationship between MMP levels and mortality

Compared with surviving patients, baseline levels of MMP-2 were significantly higher
in patients who died during follow up (Table 2). Stratification by causes of death also demonstrated that higher levels of MMP-2
were found in patients who later died from circulatory disease, but higher levels
of MMP-8 were found in patients who died from respiratory disease. Baseline MMP-1
levels were lower in patients who later died from malignant disease, but this association
disappeared after adjusting for age, sex and disease duration (data not shown). The
association of high baseline MMP-2 levels with patients who later died also disappeared
after adjusting for age, sex and disease duration. However, the association of high
MMP-8 levels with patients later dying of respiratory disease remained after such
adjustment (P = 0.01).

We next investigated whether the mortality risk increased with increasing levels of
MMPs. Separate Cox analysis for each MMP, with adjustment for age, sex and disease
duration demonstrated significant increases in the hazard ratio (HR) with increasing
levels of MMP-8 and MMP-9 (Table 3). No association with MMP-2 levels was seen in Cox models adjusted for age. Analyses
of cause-specific mortality demonstrated significant associations of MMP-8 and MMP-9
with mortality due to respiratory disease (Table 4). No associations were found with any MMPs for deaths due to circulatory disease
or neoplasia.

Multivariate survival analysis

In stepwise Cox proportional hazards regression models that included all MMPs, only
MMP-8 was found to be significantly associated with all-cause mortality (HR = 1.006
per ng/ml, 95% CI 1.002, 1.01, P = 0.003). Further multivariate analyses including other risk factors revealed that
MMP-8 level was predictive of mortality independently of age, smoking at baseline,
nodular disease, and taking CVD drugs at baseline, all of which were associated with
all-cause mortality (Table 5). Other disease activity and severity markers (CRP, ESR, HAQ, RF, Larsen score) were
excluded due to non-significance in stepwise analysis (Table 5).

Mortality due to circulatory diseases was not associated with levels of any MMP, although
when mortality due to heart disease alone was analysed a significant association with
baseline MMP-9 levels was found (HR 1.0013 per ng/ml, P = 0.01). This remained significant (P = 0.01) after adjusting for smoking at baseline and nodular disease, but significance
was lost when adjusted for patients taking CVD drugs at baseline. The latter association
was highly significant (HR 4.60, 95% CI 2.39, 8.94, P < 0.0001). As in the case of all-cause mortality, we found that age, smoking at baseline
and nodular disease were all significantly associated, with or without adjustment
for taking CVD drugs.

Mortality due to neoplasia was not associated with levels of any MMP. The most significant
predictors were older age, and smoking at baseline (data not shown). Multivariate
analysis of mortality due to respiratory disease showed that MMP-8 level was a highly
significant predictor along with age, nodular disease and presence of RF (Table 5). Although significant in univariate analysis, MMP-9 levels were not significantly
associated in a model that also included MMP-8 levels. Smoking and measures of inflammation
were not associated. Separate analyses of patients who had died from pneumonia or
from other causes of respiratory disease demonstrated a significant association with
MMP-8 levels in both cases, although the number of patients who had died from causes
other than pneumonia was small (n = 13) (Table 6).

Table 6. Multivariate baseline predictors of mortality due to pneumonia and mortality due to
other respiratory diseases in rheumatoid arthritis patients

Figure 1.Kaplan Meier survival curves for RA patients showing the relationship between baseline
serum levels of metalloproteinase (MMP)-8 and mortality due to respiratory disease
or pneumonia alone. (A) Respiratory disease. (B) Pneumonia alone. For these analyses, the levels of MMP-8 were divided into those
below (<) or equal to and above (≥) the median (20.07 ng/ml).

Discussion

Our data indicate that the serum level of MMP-8 (neutrophil collagenase) is a strong
predictor of mortality in RA, especially that due to respiratory disease. As far as
we are aware, this is the first study to identify a biomarker that is predictive of
respiratory disease-related mortality in RA. Age, nodular disease and presence of
RF were also predictive but the MMP-8 association was independent of these. Although
the majority of deaths in this group of patients were due to pneumonia, separate cause-specific
analyses revealed that MMP-8 levels were associated with mortality from both pneumonia
and other forms of respiratory disease. The latter included pulmonary fibrosis, COPD,
bronchitis, and bronchiectasis, but these were analysed as a single group since the
numbers were too small to investigate specific sub-groups. Further research on larger
cohorts of patients will be needed to determine whether MMP-8 levels are predictive
of mortality from specific respiratory diseases other than pneumonia.

Apart from MMP-8 and age, the other baseline parameters most strongly associated with
mortality in this study were smoking, (all-cause mortality, cancer mortality), taking
CVD drugs (all cause-mortality, cardiovascular mortality), nodular disease (all-cause
mortality, respiratory disease mortality), and RF (all-cause mortality, respiratory
disease mortality). The findings on all-cause mortality are consistent with previous
studies in RA. Although we and others have shown that higher levels of MMP-8 are associated
with smoking in RA and other conditions [31,32], our results suggest that the association of MMP-8 with all-cause and respiratory
disease mortality is independent of smoking.

It is interesting that none of the other MMPs examined here were associated with mortality
after adjusting for MMP-8 levels or age, sex and disease duration, even though all
of the MMPs, apart from MMP-2, showed significant correlations with inflammatory and
disease severity markers, and the literature on non-RA populations has reported associations
of MMP-2, MMP-3 and MMP-9 levels with CVD [19-24]. In this study we found that the baseline levels of MMP-2 were significantly higher
in patients who subsequently died in the follow up period, but the association disappeared
after adjusting for age. This can be explained by the significant association between
age and MMP-2 levels seen in the correlation analyses. Although we found no association
of MMP levels with mortality from circulatory diseases in general, we did find that
MMP-9 levels were associated with mortality specifically due to heart disease. This
association disappeared after adjusting for patients taking CVD drugs at baseline.
This can probably be explained by our data showing that these particular patients
had significantly higher levels of MMP-9, and is consistent with previous studies
in no-RA populations showing higher levels of MMP-9 in heart disease [19-21,24].

The mechanisms behind the association of MMP-8 levels with mortality from respiratory
disease are likely to involve the tissue destructive effects of this MMP. Several
cell types may contribute towards the levels of MMP-8 but the major source is most
likely to be activated neutrophils. Many previous studies have demonstrated the likely
role of neutrophil-derived MMPs in the pathogenesis of respiratory diseases, including
acute respiratory distress syndrome or acute lung injury [33,34], COPD [35,36], cystic fibrosis [37], interstitial lung disease [38], idiopathic pulmonary fibrosis [39] and bronchiectasis [40,41]. Furthermore, pulmonary MMP concentrations have been shown to be elevated in patients
with hospital-acquired pneumonia [42,43]. All of these conditions are characterized by an influx of neutrophils into sites
of inflammation and subsequent pulmonary tissue injury, the severity of which is associated
with MMP levels [39,40,42,43].

It is interesting in this study that measurement of MMP-8 levels at a single time
point was predictive of long-term mortality, even though in many cases the levels
were measured years before death. One possibility is that elevated MMP-8 levels may
reflect chronic ongoing neutrophil activation, which may persist for many years in
RA. Peripheral blood neutrophils in RA patients have been reported to be primed but
show functional impairment in Fc-mediated generation of reactive oxygen species [44]. This has been suggested to account for the increased susceptibility to bacterial
infection in patients with severe RA and may explain the increase in respiratory diseases
such as pneumonia. The presence of high-risk bacteria in patients with hospital-acquired
pneumonia has been shown to be associated with significantly higher MMP-8 and MMP-9
levels and activity in the bronchoalveolar lavage fluid [42,43]. It has also been reported that artificial ventilation may further promote protease
activation [43,45].

There are a number of possible limitations to the present study. Apart from determining
which patients were taking CVD drugs, other comorbidities were not recorded at baseline.
Various comorbid conditions may be associated with elevated levels of MMPs, so these
may represent unmeasured confounders. The information on patients taking drugs for
CVD problems allowed us to provide an estimate of patients suffering from cardiovascular
morbidity at the time of recruitment [46]. However, this may have been and under- or overestimate of the amount of cardiovascular
morbidity present since some patients with CVD may not be taking any specific CVD
drugs or may have stopped them (for example after myocardial infarction. Others may
have been taking certain drugs as a preventative measure because of increased cardiovascular
risk. Nonetheless our data show that patients on CVD drugs at baseline were significantly
more likely to die from all-cause mortality, and heart disease in particular, than
patients not on these drugs. Our data also show that patients taking CVD drugs at
baseline had higher levels of MMP-2, 8 and 9 than patients not taking these drugs.
However this does not explain the increased risk associated with elevated MMP-8 level,
since this was associated with all-cause mortality independent of taking CVD drugs,
and the latter were not associated with mortality from respiratory disease in models
in which MMP-8 levels were highly significant.

A second possible limitation is the generalisability of the findings to the wider
RA community since the patients studied were hospital-recruited and the study population
was likely to include patients with more severe disease. The treatment of patients
at baseline and during the follow up period also underwent changes in standard UK
practice for treatment of hospital-based RA patients over the period 1993 to 2011.
This would invariably have led to different treatment regimes based on the severity
of disease at baseline, and during follow up. It is possible that different treatments
may have resulted in different effects on MMP levels, but it was not possible to control
for this in this study. Previous studies have indicated that the use of oral steroids
is associated with increased morbidity and mortality from lower respiratory tract
infections [12,47], and we have found in a separate, previously described population of well-characterised
RA patients [48] that those taking oral steroids have significantly higher levels of MMP-8 and MMP-9
than those not taking these drugs (unpublished observations). We have also confirmed
in this second population that MMP-8 levels are associated with mortality from pneumonia,
but this was independent of steroid use, which did not demonstrate a significant association
(unpublished observations).

The interpretation of death certificate data is another potential limitation to the
current study, especially with regard to deaths from pneumonia or chest infections
when the associated or contributing cause (for example, stroke) may in fact be the
main cause. However, in the current study the majority of patients dying from pneumonia
(n = 23/29) had this as the only cause, or the primary cause alongside RA as an associated
cause. These patients also demonstrated a highly significant association of MMP-8
levels with mortality, similar to the total group who died from pneumonia (data not
shown).

Another possible limitation was the measurement of MMP levels at a single time point,
and cumulative measures of MMP-8 levels during follow up may possibly have provided
better predictive information. Importantly the association of MMP-8 levels with mortality
was independent of traditional measures of inflammation. In the present study, single
measurements of ESR and CRP at study entry were not generally predictive of mortality
in models that also contained MMP-8 levels.

Conclusion

Our results indicate that in patients with established RA, high serum levels of MMP-8
are predictive of respiratory disease-related mortality, and provide additional predictive
information on mortality in RA, which is not provided by traditional measures. These
results also point to MMP-8 as a possible therapeutic target to reduce protease-mediated
damage in pneumonia and other respiratory diseases in patients with RA.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

DLM and NBN carried out the biomarker measurements. DLM carried out the statistical
analysis. NBN and PTD participated in the design of the study, and recruitment of
patients. DLM conceived the study, participated in its design and coordination, carried
out analysis and interpretation of data, and drafted the final manuscript. All authors
read and approved the final manuscript.

Acknowledgements

We wish to thank Mrs June Fisher and Mrs Sheila Clarke (metrologists) for their help
with data collection. This work was supported by the Haywood Rheumatism Research and
Development Foundation.